Antiphospholipid antibodies are a heterogeneous family of immunoglobulins that includes, among others, lupus anticoagulants and anticardiolipin antibodies. Lupus anticoagulants (LA) behave as acquired inhibitors of coagulation, prolonging phospholipid-dependent in vitro coagulation assays (Mackie, J. I., et al., (2000) Antiphospholipid Syndrome, London, UK: Springer; pp. 214-224), whereas anticardiolipin antibodies are measured by immunoassay, utilizing cardiolipin or other anionic phospholipids in solid phase (Loizou, S., et al., (1985) Clin. Exp. Immunol. 62: 739-744). Despite their name, antiphospholipid antibodies do not recognize phospholipids, but instead recognize plasma proteins bound to suitable anionic, but not necessarily phospholipid, surfaces. Among these, β2-glycoprotein 1 (McNeil, H. P. et al., (1990) Proc. Natl. Acad. Sci. USA 87: 4120-4124; Galli, M. et al., (1990) Lancet 334: 1544-1547) and prothrombin (Arvieux, J. et al., (1995) Thromb. Haemost. 74: 1120-1125) are the most commonly investigated antigenic targets. Most anticardiolipin antibodies require β2-glycoprotein 1 (β2-GP1) to react with cardiolipin in immunoassays (Galli, M. et al., (1990) Lancet 334: 1544-1547).
Specific subgroups of anti-β2-GP1 (Galli, M. et al., (1992) Thromb. Haemost. 68: 297-300) and antiprothrombin antibodies (Galli, M. et al., (1997) Thromb. Haemost. 77: 486-491) are responsible for the lupus anticoagulant activity in phospholipid-dependent coagulation tests. Two forms of antiphospholipid syndrome (APS) have been described: a “primary” syndrome (Asherson, R. A., et al., (1989) Medicine 68: 366-374), with no evidence of an underlying disease, and a “secondary” syndrome (Alarcon-Segovia, D. et al., (1989) Medicine 68: 353-365), mainly in the context of systemic lupus erythematosus (SLE). Thromboembolytic events are reported in approximately one-third of antiphospholipid-positive subjects. However, the results of clinical studies are largely influenced by factors such as differences in study design and eligibility criteria, and by the diversity of antiphospholipid antibodies in terms of types, isotypes, cutoff, and laboratory methods employed for their detection.
Primary APS is a pathological hemostatic condition characterized by unexplained thrombosis, recurrent fetal loss, thrombocytopenia, and/or neurological disorders. Secondary APS occurs when antiphospholipid antibodies are present in subjects with other autoimmune disorders such as SLE, as originally described by Conley and Hartmann ((1952) J. Clin. Invest. 31: 621-622). The development of antiphospholipid antibodies may also result from the administration of drugs such as chlorpromazine, procainamide, thorazine and other medications. LA autoantibodies are directed against heterogeneous complexes of anionic phospholipids (e.g. cardiolipin, phosphatidylinositol, phosphatidylethanolamine and phosphatidylserine) (Thiagarajan, P. et al., (1980) J. Clin. Invest. 66: 397-405) and phospholipid-binding proteins (Triplett, D. A. (2002) Arch. Pathol. Lab. Med. 126(11): 1424-1429) in plasma. The major protein components of the LA autoantigens include β2-GPI, prothrombin and annexin V. LA antiphospholipid antibodies are characterized by their ability to prolong in vitro clotting times in coagulation-based assays such as phospholipid-sensitive aPTT (active partial thromboplastin time), kaolin clotting time, dilute Russell's Viper Venom Time test (e.g. DVVtest®, American Diagnostica Inc.) and dilute prothrombin time (dPT) tests (Thiagarajan, P. et al., (1986) Blood 68: 869-874; Exner, T. et al., (1990) Blood Coag. Fibrinol. 1: 259-266).
The aPTT assay is a kinetic assay that measures the recalcification time of plasma. By activating the plasma to a maximum level before clotting can occur, aPTT is used to screen subjects with a bleeding tendency for deficiencies in coagulation factors involved in the intrinsic pathway (Factor VII and Factor XIII excluded) and to determine the presence of a non-specific inhibitor, such as an LA. The aPTT assay is also used to evaluate the effect of therapy and to monitor and regulate heparin therapy. In this assay, kaolin (powdered silica, Celite or ellagic acid) is added to the plasma for approximately 3 minutes, depending on the activator, at 37° C. to activate it, after which partial thromboplastin (cephalin or soya phosphatide) and calcium chloride are added to induce clotting. Prolongation of aPTT can be caused by a deficiency in one of the clotting factors involved in the intrinsic pathway, especially Factors XII, XI, X, IX, VIII, V, II and I. Prolonged aPTT is seen after a massive blood transfusion, heparin therapy, hemophilia A, acquired Factor VIII inhibitor, antiphospholipid antibodies, over-anticoagulation with coumarins, heparin therapy, or an error in specimen collection. When a prolonged aPTT is observed, more specific single factor assays and mixing studies are performed to identify its exact cause.
Clinical studies show that a dPT is an effective antiphospholipid coagulation assay and can identify antiphospholipid antibodies that are not detected in other assays (e.g. phospholipid-sensitive aPTT and dRVVT)(Liestol, S. et al., (2002) Thromb. Res. 105: 177-82; Triplett, D. A. et al., (1983) Am. J. Clin. Path. 79: 678-682; Arnout, J. et al., (1994) Br. J. Haematol. 87: 94-9). The addition of a dPT assay to the antiphospholipid antibody testing panel has been shown to increase the sensitivity of detecting LA in subject samples (Mackie, J. I. et al., (2004) Thromb. Res. 114: 673-674).
ACTICLOT® dPT™ (American Diagnostica Inc.) is an example of a fully integrated dilute prothrombin time diagnostic kit for screening and confirming the presence of phospholipid-dependent LA autoantibodies for the definitive diagnosis APS. The ACTICLOT® dPT™ activator used for the screening protocol contains a unique formulation of lipidated recombinant tissue factor and calcium. The ACTICLOT® LA Phospholipid reagent used in the confirmatory protocol contains a unique formulation of phospholipids specifically designed to demonstrate the phospholipid-dependent nature of the LA's detected in samples that are positive in the ACTICLOT® dPT™ screening protocol.
A commonly used protocol is the combined DVVtest® and DVVconfirm® assay (American Diagnostica). DVVtest® is a dilute Russell's Viper Venom Time (dRVVT) assay intended for the determination of LAs in subject plasma. DVVconfimm®is intended to confirm the presence of LAs in plasma that tested positive using the DVVtest®. All DVV test reagents normally used for screening contain Russell's viper venom (RVV) as the active component. DVV test reagents containing RVV directly activate Factor X to Factor Xa in the presence of a low level of phospholipids and calcium, converting fibrinogen to fibrin and leading to detectable clot formation in plasma. This direct activation bypasses the Contact and Intrinsic Factors in the coagulation cascade, thereby excluding interference from deficiencies of Factors VIII, IX, XI, and XII, or their respective inhibitors.
DVV confirmatory reagents contain Russell's viper venom and a high amount of several phospholipids. The screening of plasma involves mixing DVV test reagents with subject plasma and determining the clotting time. A prolonged clotting time relative to normal plasma is indicative of the presence of LA. If the plasma contains LA, the plasma is then retested with the DVV confirmatory reagent and should have a significantly reduced clotting time relative to that with DVV test reagent. The reduced clotting time with DVV confirmatory reagents is due to high phospholipids in the DVV confirmatory reagent that neutralize the LA autoantibodies.
One problem with this approach is that coagulation-based test and confirmatory reagents must be matched to one another to obtain a positive result. This is because both reagents contain phospholipids and a procoagulant activator reagent, which initiate clot formation. If the test reagent and confirmatory reagent are not matched correctly, then false positives or false negatives can be obtained. This also makes manufacturing more difficult. During manufacturing of the reagents, the activity of the active components may change, which makes it difficult to properly match the two test and confirmatory reagents.
Due to the heterogeneous nature of the pathological phospholipid-dependent autoantibodies, it is widely recognized that no single LA coagulation assay identifies all LA antibodies. In 1995, the International Society on Thrombosis and Haemostatis (ISTH) Scientific Subcommittee on Antiphospholipid Antibodies recommended that each plasma sample suspected of containing LA should be tested in at least two LA diagnostic assays to increase the probability of identifying LA (Liestol, S. et al, (1983) Thromb. Res. 105: 177-182). Additionally, the ISTH SSC recommended that a definitive diagnosis of LA also require demonstration of the phospholipid-dependent nature of the autoantibodies. This is accomplished by performing a second confirmatory coagulation assay in the presence of high amounts of phospholipids. The hallmark of the presence of LA is a significant reduction of clotting time of the high phospholipid confirmatory assay as compared to the low phospholipid-screening assay.
Using the same active reagents in both the testing/screening and confirmatory steps of a coagulation-based LA assay, therefore, would be a significant advance in the art to prevent the problems of these testing protocols. Furthermore, a common reagent useful in all three LA coagulation-based assays would significantly decrease the time and labor involved in diagnosing and/or monitoring disorders characterized by the presence of antiphospholipid antibodies.